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Description
Human CDK4 ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant and centrifuge at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. Cell Supernatant: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and mince the tissue. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and the supernatant can be assayed. Preparation before testing: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the gradient working solution of the standard: Add 1 mL of universal diluent to the lyophilized standard. Let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water (concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C. 2. Sample addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate. This will reduce the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculating experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a cyclin-dependent kinase 4 (CDK4) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of cyclin-dependent kinase 4 (CDK4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Cyclin Dependent Kinase 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Cyclin-dependent kinase 4, also known as cyclin-dependent kinase 4 or cell division protein kinase 4, is an enzyme encoded by the CDK4 gene. CDK4 is a member of the cyclin-dependent kinase family. The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This protein is highly similar to the gene products of S. cerevisiae cdc28 and S. pombe cdc2. It is a catalytic subunit of a protein kinase complex and is important for progression through the G1 phase of the cell cycle. Its activity is restricted to the G1-S phase and is controlled by the regulatory subunits D-type cyclin and the CDK inhibitor p16INK4a. This kinase has been shown to be responsible for phosphorylation of the retinoblastoma gene product (Rb). Mutations in this gene and its related proteins, including D-type cyclin, p16 (INK4a), CDKN2A, and Rb, have been implicated in the tumorigenicity of various cancers. A specific point mutation (R24C) in CDK4 was first identified in a melanoma patient. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, cell supernatant, tissue homogenate, etc. |
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4.1 ★★★★★
Based on 26 reviews
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Product Reviews
★★★★★ 5
Truly Chew Proof (so far)
Size: Yellow
My dog loves this corn thing (he is 1 year old and roughly 50 lbs). He has gotten to be an aggressive chewer, but this has been one of the only toys that has kept up after long use (4 months since I bought it) and not been destroyed in minutes. There is a nice squeak to it, and I really have to press hard to get it going, but it is easy for him to do it. It is easy to clean, too, because it is rubber.
My only comment is for owner awareness: the description says "good for teeth and keeps them clean". This is false. I believe no dog toy or kibble will "clean" their teeth. You, the owner, just have to keep up with their oral hygiene through brushing. As for the other claims -- the training, weight management, and preventing chewing furniture -- again, use your best judgment. I did not purchase the toy for those claims. But as a durable chew toy, this yellow corn does the job and satisfies the natural instinct to chew.
Note: Some of the negative reviews for durability are for the alternative blue rubber bone, which I did not purchase.
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Reviewed in the United States on April 22, 2025
★★★★★ 5
Highly recommend
Size: Blue
Update! My puppers has had this for months now and I wash it every now and then and its so durable!
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Reviewed in the United States on June 11, 2026
★★★★★ 5
The best chew, play and teeth cleaning toy
Size: Blue
I have a 24 lb medium australian labradoodle multigen. She loves to chew and nothing soothed her teething like this toy. It also cleans the adult teeth that came in. So win win. The squeakers didn't last but that was my fault for squeaking it to tire her out in play. We personally love this toy and will purchase it again when it wears out. So thanks for a great product. 😉
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Reviewed in the United States on March 23, 2025
★★★★★ 4
Not indestructible, but pretty close!
Size: Yellow, Size: Yellow
I adopted a pit mix a little over a month ago, and so far he's managed to destroy every single toy he's had, including ones meant for aggressive chewers. He's a sucker for a good squeaky toy, and this had good reviews (and was affordable) so I decided to check it out.
He's had his corn for a week now, and surprisingly, it's still holding up pretty well - though there are some caveats to that.
My boy didn't even want to wait for me to take his corn out of the plastic bag, he too excited to get it. He went to town immediately, to the point where he was sitting in a puddle of his own drool after about an hour. He managed to squeak it (which I couldn't do myself), but still hasn't really gotten the hang of doing so - and the squeaker, when wet, does sound a little... Well, wet.
He managed to have bite marks in it within the first six hours. Within a day, there were tiny bits of yellow rubber starting to appear anywhere that he played with this toy. A week later and there are some decent sized chunks missing from the corn - some of which I've had to cut off when they got loose and I worried about him choking on them.
He loves his corn cob, and will likely get at least a few more weeks of play out of it before I have to toss it for his safety. I'm unsure of whether the squeaker is still functional, but he seems to love it whether or not it squeaks.
Overall, it's not a bad toy for most aggressive chewers, but it will not last forever. The rubber is as heavy as the regular red Kong toys, but it feels slightly softer, which might contribute to his ability to break off little pieces.
Make sure you keep an eye on your dog with this toy, or with any other, especially if they are chew goblins. But I'd definitely buy this toy again.
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Reviewed in the United States on October 6, 2024
★★★★★ 5
Solid piece
Size: Yellow
Border Collie loves it. Great for aggressive chewers. Prefers this to the carrot or the pineapple one. Probably better for large dogs than my medium size dog as there is some heft to it.
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Reviewed in the United States on December 25, 2025
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